Chapter 8 Flashcards
1
Q
Diabetes mellitus is
A
a disease caused by a deficiency in the production or activity of the hormone insulin (a protein), resulting in elevated blood glucose levels.
- about 5 % of all deaths r by diabetes.
- can lead to many health complications, including blindness, organ problems, limb amputations, & early death.
2
Q
Type 1 diabetes
A
- a type of diabetes caused by an inability to produce insulin
- Many with type 1 diabetes need insulin by mouth or by injection to prevent the disease from seriously
damaging their body
3
Q
Type 2 diabetes
A
- a type of diabetes caused by low insulin or an inability to use insulin
- 2 main risk factors of type 2 diabetes r a genetic predisposition & being overweight.
- North America = type 2 diabetes is rising –> Canadians eat more refined foods & sugar, & less natural foods & fibre, than ever before.
- more people r suffering from insulin resistance, obesity, and type 2 diabetes.
4
Q
Insulin was isolated by which 2 researchers?
A
Dr. Frederick Banting and Dr. Charles Best, at the University of Toronto in 1922
- Helen Free, born a year after the discovery, invented a method to analyze the blood sugar by dip-test urinalysis. –> (Before this, doctors tasted a patient’s urine. Sweet urine was an indicator of diabetes.)
-allowed people with diabetes to monitor their blood sugar level easily at home. These two innovations have vastly improved the lives of diabetics
5
Q
Insulin was initially collected from the pancreases of ________ & __________. What changed?
A
pigs, cows
- was allergic reactions to this insulin, even though it was necessary to keep them alive.
- Scientists tried to mass-produce acc human insulin.
- They discovered that they could insert the human insulin gene into bacteria, & the bacteria would make human insulin.
6
Q
Genetic engineering is
A
the intentional production of new genes and alteration
of genomes by the substitution or introduction of new genetic material
7
Q
Bacteria are versatile tools for genetic engineers cuz..
A
1) they reproduce quickly & often,
2) are relatively inexpensive to maintain
3) contain plasmids
8
Q
What is used to make biosynthetic human insulin?
A
- Escherichia coli (AKA E.coli) is used.
- this bacteria is very common in the human body
- E. coli transcribes & translates the piece of human DNA to make insulin, which is then harvested from the bacteria.
- Safflowers r also used to make insulin.
- After human insulin gene in the plasmid is inserted into the E. coli or the safflower, both organisms have recombinant DNA
9
Q
recombinant DNA is
A
a DNA strand that is created using DNA pieces from two or more sources (does it have to be species?)
10
Q
Restriction Enzymes
A
-1st step in genetic recombination is to isolate, or cut out, a DNA fragment that contains the desired gene
- Restriction enzymes (AKA endonucleases) occur naturally in prokaryote cells & cut DNA at specific locations
–> they act as molecular scissors
- Each enzyme recognizes a unique nucleotide sequence and cuts at one specific location & direction. –> Each restriction enzyme recognizes a specific sequence of nucleotides on a DNA strand = recognition site for that particular enzyme
- When the enzyme cuts the DNA molecule, the pieces it makes r known as restriction fragments.
11
Q
Each restriction enzyme _____________ at only 1 ______________ and in only 1 ____________. Give an example.
A
cuts (or digests), recognition site, direction
- enzyme EcoRI binds to a recognition site with the base-pair sequence 5’-GAATTC-3’ and cuts the DNA backbone between G and A.
-Notice that the recognition sites on the DNA are palindromic when you consider both strands
-Another EcoRI enzyme makes the same cut in the complementary DNA strand. - This leaves only a small # of the H-bonds holding the DNA molecule together, allowing the DNA molecule to be easily separated, resulting in complementary “sticky” ends.
12
Q
A restriction enzyme is
A
- AKA restriction endonuclease
an enzyme that cuts DNA at a specific location in a
base sequence
13
Q
A recognition site is
A
a sequence of bases on a DNA strand that restriction enzymes bind to.
14
Q
A restriction fragment is
A
a fragment that is produced when a DNA strand is cut by a restriction enzyme
- Restriction enzymes r highly specific.–> Most have recognition sites for 4 to 8 base pairs.
–> The fewer base pairs that they need for the recognition sequence the more cuts that are made in a DNA strand.
–> In a random DNA sequence, the probability of
finding a particular four-pair sequence is 1 in 4^4
( 1/256 or 0.4 %).
–> The chance of finding the six-pair sequence that EcoRI needs to perform a cut is 1 in 4^6 (1/4096 or 0.02 %).
15
Q
2 possible outcomes result from a restriction enzyme cutting a DNA molecule.
A
- If cuts r made straight across the strand, blunt ends r created.
- If cuts are made in a zigzag, sticky ends r created.
- Ex EcoRI produces sticky ends, whereas SmaI
produces blunt ends - biologists prefer restriction enzymes that produce sticky ends cuz the DNA fragments that r created r easier to join to any other DNA strand that has been cut by the same enzyme.
16
Q
A blunt end is
A
the end that remains after restriction enzymes cut straight across a DNA strand; a blunt end is more difficult than a sticky end to recombine with another strand
17
Q
A sticky end is
A
the end that remains after restriction enzymes cut on a zigzag across a DNA strand; a sticky end of a DNA
fragment can form hydrogen bonds with a complementary sticky end on any other DNA molecule that has been cut by the same enzyme
18
Q
Discovery of Restriction Enzymes
A
Discovered accidentally in 1970 by Dr. Hamilton Smith at Johns Hopkins University when he was studying how bacteria resisted viral infections
- Since then, Over 2500 restriction enzymes identified, with specificity for ~200 target sequences.
~200 restriction enzymes are commercially available for molecular biology applications.
19
Q
What is the Biological Function of restriction enzymes?
A
- Protect bacteria by cutting viral DNA or RNA injected during infection.
- When a virus injects its own DNA or RNA into a bacterial cell, the bacterial restriction enzymes destroy the viral nucleic acid by cutting it in pieces
20
Q
How is a restriction enzyme named?
A
- is named after its cell of origin, plus a Roman numeral if more than one restriction enzyme has been isolated from this species.
- Ex, EcoRI (pronounced “eco-R-one”) was the first restriction enzyme isolated from E. coli,
- XhoI was the first enzyme isolated from Xanthomonas holcicola,
- HindII & HindIII were the 2nd & 3rd enzymes isolated from Haemophilus influenzae
21
Q
DNA Ligase is
A
is the enzyme that is used to join cut strands of DNA
- Works best with sticky ends but T4 DNA ligase is effective with blunt ends.
- The 2 DNA fragments must have overlapping complementary portions or be blunt ends that r properly aligned end to end
- Fragments are complementary if generated by the same restriction enzyme. –> Hydrogen bonds form between the complementary bases, but this is not a stable arrangement.
- The DNA is not fully linked until phosphodiester bonds form between the backbones of the double strands.–> DNA ligase makes this happen.
- DNA ligase facilitates a dehydration reaction, releasing H2O as phosphodiester bonds form –> Fully links DNA strands to create stable double-stranded DNA.
22
Q
Plasmids
A
- 2nd tool needed for recombinant DNA techniques
- are small circular pieces of DNA that r found in bacteria
- replicate independently of the chromosomal DNA.
- often contain genes that code for specific proteins, like those that provide resistance to antibiotics such as ampicillin or protect from the toxic effects of certain heavy metals.
- In fact, when u hear about bacteria that mutate quickly so that diseases become difficult to treat, it is often the plasmid DNA that is mutating.
23
Q
A competent cell is
A
a cell that is able to take up foreign (often plasmid) DNA from its surroundings
- Ex, E.coli
24
Q
A vector is
A
a DNA molecule that is used as a vehicle to transfer foreign genetic material into a cell. Ex, a plasmid
25
The plasmid copy number is
the # of plasmids of a specific type within a cell
- is variable & is characteristic of a particular plasmid.
- If a plasmid with a high copy # has been engineered to produce insulin, more insulin will be produced per cell cuz more copies of the insulin-producing gene are present
26
How are plasmids attached to DNA gene fragments to make recombinant DNA?
- If the target gene fragment and a plasmid are cut with the same restriction enzyme, they will have complementary sticky ends.
- The foreign fragments and the plasmid fragments can be placed in the same solution, where they anneal cuz of the complementary sticky ends.
- DNA ligase is then added to re-form the phosphodiester bonds between the fragments, resulting in a new circular piece of DNA that carries the foreign gene fragment. --> plasmid now = recombinant DNA
- The recombinant plasmid is introduced into a host cell (commonly bacteria). --> The plasmid replicates within the host cell, making multiple identical copies of the gene (gene cloning).
- The gene will now begin to express its function. Ex, if the insulin gene is in the plasmid, the host cell will begin to produce insulin
27
A restriction map is
a diagram that shows the relative locations of all known restriction enzyme recognition sites on a particular plasmid & the distances, in base pairs (bp), between the sites.
- when u add the bp up, we can see the plasmid's total length in bp
- cuz plasmids r circular, the # of fragments is always equal to the # of cuts
- allows molecular biologists to determine which plasmids & restriction enzymes might be most suitable for a particular recombo DNA procedure & to evaluate quickly the success of the cloning experiments
28
Plasmids: Transformation
- Plasmids can enter bacterial cells, replicate, and express inserted foreign genes.
- The successful introduction of DNA from another source is = transformation
- The cell that has received the DNA is said to be transformed
29
If a bacterial cell is not able to take up a plasmid that contains foreign DNA, it can sometimes be made competent in a laboratory. ELABORATE
1 is to place the bacteria in a solution that contains CaCl2 & recombinant plasmids in an
ice-water bath.
- As the bacteria cool, the calcium ions stabilize the negative phosphate ions on the phospholipid bilayer of the cell membrane.
- The CaCl2 solution is then heated quickly & re-cooled.--> sudden change from cold to hot momentarily disrupts the membrane, allowing the plasmid to enter.
- The cells r then kept at 37 °C for a period of time to stabilize & grow.
-After the cells have stabilized & grown, they r tested for ampicillin resistance. Those with a plasmid with ampicillin resistance gene grow in a medium that contains ampicillin. This means that they were successfully made competent & transformed
30
DNA hybridization
- technique of DNA hybridization is used to identify the cells that contain the introduced plasmids with the desired gene
- This gene can be identified by its unique DNA sequence cuz it will pair with a short, single-stranded complementary DNA molecule, called a hybridization probe.
- For insulin production, the probe matches a DNA segment coding for insulin (15–2500 bases long).
- A series of steps taken to identify a target DNA sequence during DNA hybridization.--> After the presence of the DNA for insulin production is confirmed, the bacteria r grown in huge quantities,
enough to produce insulin on a commercial scale.
31
Lake Sturgeon
- Lake sturgeon were once abundant in Canadian lakes but r now endangered due to overfishing for their meat & caviar.
- It is illegal to fish or consume lake sturgeon under the federal Fisheries Act.
- If conservation officers come across campers eating fish, & they suspect that the fish is not in season or is an endangered species, they can take a small sample to a forensic lab.
- If the DNA matches that of a banned species, the conservation officers have sufficient evidence to give to the police.
32
Trent University, in Ontario, has a Wildlife DNA Profiling & Forensics Laboratory
- lab tracks endangered species & supports the enforcement of the Convention on International Trade in Endangered Species (CITES).
- The polymerase chain reaction (PCR), developed in 1983 by Kary Mullis, helps them by amplifying small DNA samples for extensive testing.
- DNA fingerprinting identifies species from minimal samples, such as fish on a plate or blood on a hunting knife.
- PCR and DNA fingerprinting help analyze DNA to determine the species source, enforce conservation laws, and provide forensic evidence.
33
The Polymerase Chain Reaction is
- AKA PCR
a process that is used to make a huge # of copies of a DNA sequence in a laboratory, quickly (in a few hours) & without a host organism
- It is a simple, reliable, fast, and inexpensive method widely used in molecular biology.
- Kary Mullis, who developed PCR, was awarded the Nobel Prize in Chemistry in 1993.
- PCR focuses on replicating a specific DNA region, not the entire genome.
- The process occurs outside a cell’s nucleus, in a laboratory setting.
- The whole process takes place in a microfuge tube
(a small test tube)
34
PCR consists of three steps:
1) denaturation
2) annealing
3) elongation
- These steps r repeated in many cycles, usually 30-40
35
PCR: Denaturation
- 1st step of PCR
- a double-stranded DNA molecule is denatured, or separated, into its 2 single strands --> occurs when the DNA strand is heated to 94 to 96 °C for 20 to 40 s, breaking the H-bonds holding the 2 strands together.
36
PCR: Annealing
- 2nd step of PCR
- occurs at a lower temperature (50–65 °C) for 20–40 seconds
- Single-stranded DNA primers anneal to complementary sequences on the separated DNA strands.
- 2 primers r used, each complementary to opposite strands near the 3' ends of the target sequence. --> This results in both primers being oriented in the 5' to 3' direction toward the target sequence.
- The specific nucleic acid sequences of the primers ensure that only the target DNA fragment is amplified. --> This specificity makes PCR a highly accurate and reliable technique.
37
A DNA primer is
a short single-stranded DNA sequence, easily made in a lab, that is complementary to a sequence at 1 end of the target sequence
38
PCR: Elongation
- AKA extension
- 2 new DNA strands are made at a temp of 72 °C. --> The 2 original DNA strands, which were separated in step #1 act as templates.
- DNA pol binds to the primer-template hybrids & moves along each template, adding nucleotides that r complementary to the sequence until it reaches the 5' end of the template.
- After the 2 double-stranded DNA sequences have been made, the process can run through another cycle. --> this time 4 strands will be used as templates
& duplicated to make new copies.
- This cycle is repeated 30-40 times, resulting in a high # of copies of the specific target sequence
39
What kind of DNA polymerase is used in PCR?
- very specific DNA pol, called Taq polymerase, is needed for PCR elongation.
- Taq polymerase is from the bacterium Thermus aquaticus found in hot springs, so its enzymes can withstand high temps.
- use of Taq polymerase is vital since it is not denatured by the high temp that is used during PCR step #1.
- This would not be the case with DNA pol from a typical organism
40
By the 3rd cycle of PCR.. Within 20 cycles...
- By 3rd cycle, 2 of the 8 DNA molecules exactly match the target
molecules.
- Within 20 cycles, the PCR reaction delivers over 1 million copies of the DNA target sequence.
- Since each cycle takes only about 5 min, millions of copies of a DNA sequence can be make in < 2h
41
Gel Electrophoresis is
a method for separating large molecules, such as DNA, RNA, & proteins
- Once targeted DNA sequence has been amplified by PCR, it needs to be separated & purified from the rest of the contents in the test tube.
- A chromatography-like process = gel electrophoresis, uses the physical & chem properties of DNA to separate the fragments.
-used to find identity of an unknown DNA sample --> ex, identity of the tissues of an endangered species, like lake sturgeon, or identity of a criminal or father in a paternity test.
- Researchers may also extract a particular DNA fragment from the gel --> The extracted
fragment can be purified and analyzed to determine its exact base-pair sequence.
42
Gel electrophoresis separates nucleic acids & proteins by their rate of...
migration through a gel
- Agarose gel, made from agar (a polysaccharide from seaweed), is normally used for nucleic acids.
- DNA fragments (-ve) travel through the pores of the gel, away from a -ve electrode at the starting end & toward a +ve electrode at the destination end.
- cuz of the -ve charge that is carried by the phosphate groups, each nucleotide is ionized. ->means that nucleic acids have about the same charge-to-mass
ratio, even if they differ in length.
--> The smaller fragments of nucleotides move more
easily, & hence faster, through the pores of the gel than the longer fragments do. --> thus , DNA fragments can be separated by size. --> The smaller the fragment, the farther it travels in a given period of time.
- there are 4 steps of gel electrophoresis
43
Why is DNA attracted to the positive electrode in Gel electrophoresis?
- DNA is negatively charged, due to the phosphate groups, & is therefore attracted to a positive electrode.
44
Gel electrophoresis: Step 1
- Prepare a gel (a thin slab of agarose), & place it in a gel box between 2 electrodes.
- The gel has wells for placing the DNA samples to be analyzed.
- Add a buffer to cover the gel.
45
In vitro =
In vivo =
In vitro = experiments done in a lab or petri dish; essentially outside of the body
In vivo = refers to experiments that take place inside a living organism, such as a person, animal, or plant
46
Gel electrophoresis: Step 2
- DNA samples (e.g., PCR products) are loaded into wells in the gel alongside a lane containing DNA "marker" fragments of known sizes. -> The marker acts as a reference to determine the sizes of unknown DNA fragments by comparing their migration distances.
- A special dye=a loading dye, is added. The dye has two functions.
--> 1) cuz it is denser than the DNA, the dye keeps the DNA from floating out of the well. It sinks, along with the DNA, into the well.
--> 2) the dye travels slightly faster than the fastest nucleotide fragments. The rate at which the dye is seen to move can be used to judge the pace of the electrophoresis process
47
A molecular marker is
a fragment of a known size that is run as a comparison standard for gel electrophoresis
48
Gel electrophoresis: Step 3
- An electric current is applied to the gel, causing -ve charged DNA fragments to migrate toward the +ve pole.
- Shorter DNA fragments migrate faster & farther than longer fragments.
- Fragments of equal length travel the same distance, forming clusters which form invisible bands within the gel
49
Gel electrophoresis: Step 4
- The gel is stained after electrophoresis to make DNA fragments visible.
- Ethidium bromide is a common stain that intercalates within the DNA double-helix structure.
- Under UV light, ethidium bromide fluoresces, revealing the DNA bands in the gel.
50
ethidium bromide is
a large molecule that resembles a base pair, which allows it to insert itself into DNA; used for staining electrophoresis gels
51
DNA sequencing is
a process in which the sequence of base pairs in a DNA strand is determined
-made it possible for researchers to analyze the base
sequences of cloned genes & DNA fragments amplified by PCR
- Having complete sequence of a gene helps researchers understand how the gene functions. --> they can identify mutations, locate regulatory sequences & gene sequences, & compare homologous genes of diff species.
52
chain termination method overview
- a method for sequencing DNA
- AKA Sanger method
- by Frederick Sanger at Cambridge University (1970s)
- relies on adding labelled dideoxynucleotide (ddNTP) to a growing DNA strand. --> This nucleotide prevents the binding of the next nucleotide, thus terminating the elongation of the newly made DNA strand.
- The labels on the ddNTPs r dyes that fluoresce & can be used to identify the specific base when exposed to laser light
53
The chain termination method process
4 reaction tubes are prepared, each containing:
- DNA to be sequenced.
- DNA primer and DNA polymerase.
- Normal nucleotides (dATP, dTTP, dGTP, dCTP).
- One labelled ddNTP (ddATP, ddTTP, ddGTP, or ddCTP) in low conc. --> diff one in each tube
- Synthesis of a complementary chain makes DNA fragments of diff sizes, each with their last nucleotide labelled. --> recall that ddNTPs don't have an –OH group on the 3'C for DNA polymerase to add another nucleotide.
- contents of the 4 reaction tubes r combined & then separated by gel electrophoresis with a resolution of only 1 nucleotide. The sequence can then be determined by reading the order of the by reading the labelled ddNTPs.
- This process is now computer automated
54
What techniques for DNA sequencing were used in mapping the entire human genome?
used 2 techniques, both involving the Sanger method for DNA sequencing.
- However, one technique, called the whole-genome shotgun method, proved to be faster than the other.
55
the whole-genome shotgun method is
a DNA sequencing method that involves blowing DNA strands into many fragments & then using computer tech to sequence & reassemble the fragments
- many copies of the DNA r randomly cut into tiny
fragments by passing the DNA through a pressurized syringe.
- fragments r cloned in plasmids & then sequenced using the Sanger method.
- software is used to determine the overall sequence by analyzing the many partial sequences
- Think of tearing 10 copies of a book randomly into smaller sets of a few pages each &, by matching
overlapping pages, reassembling a complete copy of the book with the pages in the correct order.
56
Drawback of the shotgun method
If the genome has repeating sections, it is difficult (or even impossible) for the computerized analysis to detect where these repeats are.
This causes some errors in the results.
- however, newer advances in DNA sequencing tech have allowed scientists to determine the sequences of genomes at a much faster rate
57
Once the sequence of a whole genome has been derived, 2 approaches can be used for its analysis:
- structural genomics
- functional genomics
- Most research is focused on functional genomics cuz genes control the functions of cells, & thus the functions of organisms
58
structural genomics is
the study of the structure of genes & their locations in
a genome, as well as the analysis of the nucleotide sequences to locate genes within the genome
59
functional genomics is
the study of the functions of genes, the proteins they
make, & how these proteins function.
- relies on bioinformatics --> Ex, used to find a gene within a genomic sequence, align sequences in databases to determine the degree of matching, predict the structure & function of a gene product, & hypothesize evolutionary relationships for sequences
60
bioinformatics is
the use of computer tech to process a large amount of
biological data
- combo of lab experiments & sophisticated
computer analyses
61
Why r protein-coding genes important in genome analysis, and how are their functions identified?
- r crucial cuz they provide instructions for making proteins
- Once a genome= sequenced, researchers use computer algorithms to search both strands of the sequence for them. --> genome can be translated by the computer to give the amino acid sequence of the protein it could encode.
- Researchers may then assign a function to the amino acid sequence by performing a sequence similarity
search: a computer-based comparison of a DNA or amino acid sequence with databases of sequences of known genes or proteins.
- If the sequence resembles that of a previously sequenced gene, the 2 genes are related in an evolutionary sense & will likely have similar functions.
62
Analysis of genome sequencing has led to many new discoveries about genetic organization. List 3
1) eukaryotic genomes sequenced to date contain large # of previously unknown genes—many more than scientists expected to find. --> Ex, in Caenorhabditis (a nematode)12 000 of 19 000 genes have no known function. --> Identifying these genes & their functions is one of the major challenges for molecular geneticists
2) the degree to which different organisms, some widely separated in their evolution, contain similar
genes --> Ex, tho the yeast Saccharomyces is separated from our species by 100s of millions of years of evolutionary history, about 2300 of its about 6000 genes r related to mammalian genes.
- Many of these related genes control progress through the cell cycle. The similarities r so close that the yeast & human versions of several genes can be interchanged with little or no effect on cell functions in either organism.
3) Gene sequences show that eukaryotic genomes contain large # of non-coding sequences, most of them in the form of repeated sequences of various lengths & numbers. --> tho these sequences make up 25 to 50 % of the genomic in diff eukaryotic species, their functions r unknown at this time
63
Nanopore Sequencing
- a revolutionary method that scientists have been working to perfect since 1995
- drawing individual strands of DNA through tiny sub-microscopic holes = nanopores & reading the DNA sequence, one bp at a time
--> All of the DNA bases (A, C, T, and G) fit precisely through a 2.5 nm hole.
- Bases are identified by measuring variations in electrical current as they pass through the nanopore.
- Nanopores can be made from transmembrane proteins or silicon sheets with drilled holes.
- The development of nanopore sequencing is one of many efforts by genetic technologists around the world to win the “$1000 genome initiative.” --> The race is on to come up with a method that will allow any human to have his or her entire genome sequenced for a price.
64
A DNA microarray is
- AKA gene chip
a solid surface that has a microscopic grid of many DNA fragments, called probes, attached; used
to determine gene expression
- designed to hold many individual DNA samples,
ranging from just 10 to 2.1 million --> These DNA samples= probes r known sequences, & the location of each probe on the chip is also known.
- The tech used to create microarrays is similar to the that used to lay out electronic circuits on a computer chip.
- helps pinpoint the functions of genes, rather than just locations, & to compare the expressions of genes in diff types of cells or diff organisms
- can be used to understand the processes involved in the development of an organism or to detect genetic mutations by comparing an individual’s DNA with "normal" DNA
65
How does microarray technology work?
- starts with isolation of mRNA from the 2 types of cells to be compared: a reference cell (a normal cell) & an experimental cell (such as a cancer cell).
- The extracted mRNA is used to build complementary DNA (cDNA) libraries.
- cDNA from each cell type is labelled with distinct dyes (e.g., green for normal, red for cancer).
- The cDNA sequences r then denatured into single strands & placed onto the surface of the DNA microarray
- DNA microarray has probes that correspond to the coding genes of the genome being studied.
--> Any labelled cDNA that is complementary to the sequence of a probe will bind (hybridize) to this probe, while cDNA with no matching sequence will be washed off.
-The fluorescence is detected, & its location is recorded.
--> Green spots= the reference cDNA was hybridized with the probe
--> Red spots=the experimental cDNA was hybridized with the probe; in other words, the gene was
active in the experimental cell (the cancer cell).
-->Yellow spots= cDNA for both the reference cell & the experimental cell were hybridized. (red + green light= yellow light) Both the normal cell &
the cancer cell express the same gene.
-Researchers can determine which genes
have altered expressions in the cancer cell.
- advantage of the microarray is that the expressions of 1000s of genes can be identified simultaneously in a particular cell.
66
“heirloom varieties.”
- Farmers prioritize crops and animals that are easy to grow, store, and ship, pest-resistant, and high-yielding.
- As a result, some varieties of agricultural plants & animals r no longer planted or raised
- in Canada & other countries, there's a movement to raise & protect these “heirloom varieties.”
-->An heirloom variety=a variety of a species that was grown for a long time but is no longer commercially produced. --> Ex, gardeners raise heirloom tomatoes & hobby farmers raise endangered horse populations
67
Why is maintaining heirloom variety population important?
- Maintaining production of these varieties preserves the genetic diversity of their species.
--> Ex, If a disease suddenly wipes out cabbage crops, 1 or more of the heritage varieties might be resistant to this disease.
- Agronomists would breed these plants & breed them with other cabbage plants to give their disease resistance to all cabbage plants. --> they might extract the gene for disease resistance from the healthy plants & insert it into other cabbage plants.
68
Biopharming is
a process in which genetically engineered host organisms r used to make pharmaceuticals or other
products that are useful to humans
- Some methods involve the genetic engineering of organisms to make a target protein.
--> Some genetically modified goats r able to
produce spider silk proteins in their milk.--> These proteins can be removed from the milk & used in manufacturing
- The change is engineered in the germ line of the species, & the ability to make the new protein is passed on to the offspring of the og genetically engineered goat.
- Medically vital proteins, like life-saving anticoagulant antithrombin, can be make inexpensively by genetic engineering, relative 2 lab techniques.
- , it should be approached with caution. --> Even a minor change in an animal’s genome may prove to have negative consequences in the future, such as to an animal’s long-term health.
69
A transgenic organism is
- AKA genetically modified organism, GMO
an organism that has been modified to carry genes it
does not normally carry --> been changed by scientists to contain one or more genes from another organism
--> Ex, The E. coli bacteria altered to become insulin factories are GMOs.
70
More recent developments in insulin production may be even cheaper & faster than the bacterial methods discussed earlier. ELABORATE
- genetic engineers at University of Calgary discovered how to get safflowers to produce insulin.
- The DNA is easily manipulated, & the desired product(s) can be harvested in the oil from the seeds.
- Safflower is an easily grown thistle-like plant that prefers arid conditions.
- Maurice Moloney = 1of the scientists behind the engineering of safflower-produced insulin. -->At an agricultural biotechnology conference in Saskatoon, Saskatchewan, he explained how plants could be used to produce a biodegradable plastic called polyhydroxybutyrate (PHB):
71
polyhydroxybutyrate (PHB)
is a biopolymer produced in certain bacteria.
- PHB production requires the intervention of 3 enzymes. --> Plants lack these enzymes, but the bacterial genes specifying these biochemical conversions have been added.
- Results in a significant amount of carbon metabolism re-routed into making PHB.
- One of the main factors limiting the wide use of PHB polymers as an environmentally friendly plastic is the cost of production in microorganisms.
- Production in plants could address this problem & render PHB an economically viable material a variety of disposable packaging
72
Why did scientists not stick with bacteria to produce insulin & other genetically engineered products? Why have they researched the use of goats & plants as protein factories?
1) Economic Advantages: Producing proteins in animals & plants is often cheaper than maintaining bacterial production in controlled laboratory conditions. --> Ex, Cheaper plant-based insulin (e.g., from safflowers) could improve accessibility in developing countries, addressing global diabetes-related deaths (rmr 5% worldwide=diabetes).
2) plants and animals may be better to use than bacteria cuz larger organisms can produce larger molecules.--> Ex, Transgenic goats can make larger
proteins, with more complex folding patterns, than bacteria can.
3ish)--> Other transgenic organisms r engineered to be bigger and/or better versions of themselves, which is usually considered to be a benefit commercially.
73
Transgenic Plants: Canola, Advantages & disadvantages
- Genetically modified (GM) canola is engineered to resist specific herbicides, allowing effective weed control without harming the crop.
- The oil produced from GM canola is identical to that from conventional varieties.
- ~80% of the western Canada canola crop is the genetically modified variety
- Farmers using GM canola spend ~40% less on herbicides & get up to a 10% yield increase.
- GM crops have the potential to combat hunger by offering higher yields and pest resistance, especially in developing countries.
- Patented GM seeds=expensive, limiting access for some farmers, despite their benefits in improving food security for impoverished families.
74
Knockout Mice
- Genetically engineered mice with 1 or more genes turned off to study gene purpose & effects.
- r not acc “knocked out.” --> they're simply homozygous for 1 particular gene. Both copies of this gene r altered to a non-functional state so that scientists can study the effects on a mouse without it.
- r used to study the purpose of each gene & to draw parallels to humans. --> have been very useful in learning about the effects of certain genes on human health, cuz mouse genome is similar to human one.
--> Ex, in 1 mouse, scientists knocked out the p53 gene, the gene that helps stop tumour growth.
- Scientists discovered that humans r more likely to get certain cancers if they have a mutation in this gene.
--> p53 knockout mice got cancers but not in exactly the same tissues as humans with the mutation do. --> useful research is accomplished with this method,
even though the expression of the genes may vary
75
Concerns about GMOs
- many are concerned that there may be far-reaching irreversible effects
1) GMOs like herbicide-resistant crops (ex, GM canola) could outcompete native species, potentially becoming invasive weeds due to herbicide resistance-->not proven, but the Canadian government & other governments in European Union=concerned.
--> Transgenic fish have escaped &mixed with wild species. While the transgenic fish had mating advantages over the wild species, the offspring of the transgenic fish were not as viable as the wild fish offspring. --> Thus, the transgenic fish reduced the population of the fish species overall.
2) crops/animals that make med products may interbreed with native plants/animals due to proximity--> a concern cuz of potential cross-pollination and contamination.
3) GM plants might contain toxins & harm any animals that eat them. Ex, some medicine-producing safflowers= poisonous.
4) Intensive herbicide use on GM crops could pollute soil and water, impacting ecosystems. --> GMOs
often advertised as reducing need for pesticides but its hard to make general statements cuz each plant species reacts differently under diff conditions.
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Regulations vary a great deal, depending on the country developing the GMOs. Elaborate
- Rigorous testing in North America & Europe delays GMO approval by up to 10 years.
- Faster releases in countries like China risk environmental consequences due to limited testing.
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Economic and Ethical Issues of GMOs
- Patents on GMO seeds restrict farmers from saving seeds, increasing dependence on biotech companies.
- Legal disputes over patented seeds raise questions about patent laws & public access to GMO technology.
- Consumer groups would like to see the Canadian Food Inspection Agency change labelling rules so that consumers can decide for themselves whether they want to buy a product that contains a GMO
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The applications of genetic engineering already include...
biofuel production, food processing, sewage treatment, drug development, & pollution control.
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Some genetic disease treatments involve a specific diet. Give 2 examples
1) PHENYLKETONURIA (PKU)
- A hereditary condition causing phenylalanine buildup, leading to brain damage, developmental issues, and seizures.
- Managed with a low-phenylalanine diet from infancy to adulthood, tho adults have lesser symptoms
- easily diagnosed in a newborn baby, using a blood sample obtained by pricking the baby’s heel
2) FAMILIAL HYPERCHOLESTEROLEMIA
- a disease that is linked to high levels of “bad,” low-density lipoprotein (LDL) cholesterol & early heart disease. --> controlled by dietary changes & a class of drugs called statins.
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Some inherited diseases r treated by directly providing the proteins that are missing due to defective genes. Give 2 examples
- Clotting proteins injected to treat hemophilia
- digestive enzymes taken orally to treat digestive disorders & cystic fibrosis (CF). CF causes build up of thick mucus in the lungs, digestive tract, & pancreatic ducts.--> prevents pancreatic enzymes from reaching the intestines.
--> Pancreatic enzyme replacement can be given to aid with digestion & food absorption.
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Surgical solutions to hereditary disorders might include repairing a defective organ or replacing it with a transplant. Give 1 example
Alpha-1 antitrypsin deficiency (AATD) can cause liver deficiency.
- the most common reason for liver transplants in children.
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gene therapy is
the insertion, removal, or replacement of genes (to correct defective genes) within an organism’s cells to treat a disease
- treats disease from the source
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Gene therapy began with experiments using ______.
mice (in 1982 i think)
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Richard Palmiter & Ralph Brinster
- 1982: Richard Palmiter & Ralph Brinster & their colleagues injected a rat growth hormone gene into fertilized mouse eggs
- The eggs were implanted into a surrogate mother, resulting in normal-sized mouse pups, some of which grew to twice the size of their littermates, gaining global attention.
- they next tried to cure a genetic growth hormone deficiency by gene therapy. --> introed a normal copy of growth hormone gene into fertilized eggs taken from mutant dwarf mice & then implanted the eggs into surrogate mothers. --> The transgenic mouse pups grew to slightly larger than normal, demonstrating that the genetic defect in these mice had been corrected. --> This type of experiment= germ-line gene therapy
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germ-line gene therapy is
a process in which germ cells (sperm cells or eggs) are
modified by integrating functional genes into their genomes
- changes made by germ-line gene therapy may be passed on to future gens
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somatic gene therapy is
a process in which therapeutic genes r transferred
into somatic cells to treat a genetic disease
- For ethical reasons, germ-line gene therapy is not allowed in humans.--> humans =treated with somatic gene therapy
- changes & effects of this therapy will be restricted to the individual & won't be inherited by offspring
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1st successful use of somatic gene therapy with a human subject was carried out in the _______. Elaborate
1990s
- by W. French Anderson and his colleagues at the National Institutes of Health (NIH)
- Subject: young girl with a genetic disorder= adenosine deaminase deficiency (ADA). --> ADA enzyme deficiency stops white blood cells from maturing, leading to a severely weakened immune system
- Children with ADA often die from infections before puberty.
- they introed a functional ADA gene into mature white blood cells isolated from the patient.
- The modified cells were reintroduced into the patient, temporarily restoring ADA enzyme function & improving immune response.
- Mature white blood cells made from stem cells in bone marrow= non-dividing & have a finite lifespan, so the therapy must be repeated every few months.
- The patient continues to receive periodic gene therapy and direct enzyme supplementation to maintain necessary ADA enzyme levels.
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Successful somatic gene therapy has also been achieved for sickle-cell disease. Elaborate
December 1998: bone marrow cells (the source of blood cells) of a 13-year-old boy were replaced with stem cells from the umbilical cord of an unrelated infant.
- hope was that the stem cells would make healthy bone marrow cells.
- procedure worked, & the patient was declared cured of the disease.
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Despite enormous efforts, human somatic gene therapy has not been the panacea that people expected. Elaborate
- have been limited advancements since the first ADA deficiency trial.
- In 1999, a teen pt died due to a severe immune response to the viral vector used in therapy.
Some children in retrovirus-based gene therapy trials to intro genes into into blood stem cells developed leukemia-like conditions.
- Somatic gene therapy is not yet effective for most human genetic diseases. --> has shown success in experimental models of genetic disorders in mammals.
- No commercial gene therapy product has been approved for humans.
- Research and trials continue to target debilitating diseases like muscular dystrophy and cystic fibrosis, as well as treatable conditions like diabetes.
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genetic screening is
biochemical or molecular tests that are used to identify inherited disorders in parents, potential
parents, embryos, or children after they are born
- can potentially allow treatment or therapy can be started before the disease fully takes hold in the body
--> ex, Huntington’s chorea= neurodegenerative disorder that doesn't affect people until after they have had children. --> People who know that this disease exists in their family can choose to have a genetic screening test to help them decide if they want to have children of their own.--> tho knowledge can be emotionally damaging
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amniocentesis is
a genetic sampling method for testing in utero
- a prenatal test that detects whether a baby carries chromosomal abnormalities such as trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), or monosomy X (Turner syndrome).
- recommended in some pregnant women, especially older ones, whose risk of mutations in the germ cell DNA is higher
- Tests r also available for phenylketonuria, cystic fibrosis, & Duchenne’s muscular dystrophy
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Human Genome Project (HGP)
- Initiated in 1990, funded by the U.S. & supported internationally to map the human genome.
- Led by Dr. Francis Collins, using the Sanger chain termination sequencing technique with a genetic landmark map approach.
-1998: Celera Genomics, led by Dr. Craig Venter, entered the race, proposing the faster shotgun sequencing technique with a three-year completion goal.
- The competition motivated the HGP to expedite their work.
- Both teams released their 1st working drafts of the human genome in Feb 2001, ahead of schedule
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8.5 & 8.6 notes
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